Lycophlegmariols A-D: Cytotoxic serratene triterpenoids from the club moss Lycopodium phlegmaria L.

Article abstract of DOI:10.1016/j.phytochem.2012.01.006

Lycopodium serratene triterpenoids, along with an abietane-type diterpene were isolated from the methanol extract of club moss Lycopodium phlegmaria L. The structures of these hitherto unknown lycopodium terpenoids were elucidated on the basis of spectros

Full text of DOI:10.1016/j.phytochem.2012.01.006

Phytochemistry 76 (2012) 117–123
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Phytochemistry
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Lycophlegmariols A–D: Cytotoxic serratene triterpenoids from the club moss
Lycopodium phlegmaria L.
Sawangjitt Wittayalai a, Supaporn Sathalalai b, Sakornrat Thorroad b, Prateep Worawittayanon c,
Somsak Ruchirawat b,c, Nopporn Thasana b,c,
⇑
a Laboratory of Natural Product, Chulabhorn Research Institute, Laksi, Bangkok 10210, Thailand
b Laboratory of Medicinal Chemistry, Chulabhorn Research Institute, Laksi, Bangkok 10210, Thailand
c Chemical Biology Program, Center for Environmental Health, Toxicology and Management of Chemicals, Chulabhorn Graduate Institute, Laksi, Bangkok 10210, Thailand
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 16 May 2011
Received in revised form 28 November 2011
Available online 24 January 2012
Lycopodium serratene triterpenoids, along with an abietane-type diterpene were isolated from the
methanol extract of club moss Lycopodium phlegmaria L. The structures of these hitherto unknown
lycopodium terpenoids were elucidated on the basis of spectroscopic analysis. Pentacyclic triterpenoids,
21b-hydroxy-serrat-14-en-3
together with four serratene triterpeneoids established as 21b,29-dihydroxyserrat-14-en-3
caffeate (lycophlegmariol A, 5), 21b,24,29-trihydroxyserrat-14-en-3b-yl dihydrocaffeate (lycophlegmari-
ol B, 6), 21 ,24-dihydroxyserrat-14-en-3b-yl 4-hydroxycinnamate (lycophlegmariol C, 7), and
14b,21 ,29-trihydroxyserratan-3b-yl dihydrocaffeate (lycophlegmariol D, 8) as well as a known lyco-
a-ol (1) and 21b-hydroxy-serrat-14-en-3
a-yl acetate (2) were isolated
a-yl dihydro-
Keywords:
Lycopodium phlegmaria L.
Lycopodiaceae
a
a
Club moss
phlegmarin (9). An abietane-type diterpene, 8,11,13-abietatriene-3b,12-dihydroxy-7-one (margocilin,
10), was isolated for the first time from a Lycopodium plant. Lycophlegmariol B (6), D (8) and compound
1 showed inhibitory effects against MOLT-3 acute lymphoblastic leukemia (T-lymphoblast) with IC50 of
Serratene triterpene
Abietane diterpene
Lycophlegmariols A–D
Lymphoblastic leukemia
14.7, 3.0 and 2.9
lM, respectively.
Crown Copyright Ó 2012 Published by Elsevier Ltd. All rights reserved.
1. Introduction
Lycopodium cernuum, showed inhibitory effects against Candida
albicans secreted aspartic protease (SAP) (Zhang et al., 2002).
Previous work reported by Shi and co-worker that lycophlegmarin,
isolated from L. phlegmaria L., showed modest growth-inhibitory
activity against human hepatoma cells BEL 7402 (Shi et al.,
2005). In this paper, the isolation and structure elucidation of three
known serratene triterpenes (1, 2 and 9) and four new serratene
triterpenoids, lycophlegmariols A–D (5–8) are described (Fig. 1).
A known 8,11,13-abietatriene-3b,12-dihydroxy-7-one (margocilin,
10) was also isolated for the first time from Lycopodium plant,
having been previously isolated from Azadirachta indica (Ara
et al., 1990). Lycophlegmariol B (6), D (8) and compound 1 (Fang
et al., 1991) showed inhibitory effects against MOLT-3 acute
lymphoblastic leukemia (T-lymphoblast) with IC50 of 14.7, 3.0
Lycopodium phlegmaria (Tagawa and Iwatsuki, 1979), also
named Huperzia phlegmaria, is one of the club mosses (Lycopodia-
ceae) found in Thailand and used as a traditional herbal medicine
(Jayaweera, 1981). The Lycopodiaceae is not only a famous source
of lycopodium alkaloids (Hirasawa et al., 2009; Ma and Gang,
2004) having acetylcholine esterase (AChE) inhibition (Liu et al.,
1986; Scarpini et al., 2003), particularly huperzine A (Ma et al.,
2006, 2007; Ma and Gang, 2008), but they are also known as a
source of serratene-type triterpenoids (Inubushi et al., 1971a,b;
Orito et al., 1972; Shi et al., 2005; Tsuda et al., 1975; Zhang et al.,
2002; Zhou et al., 2003). Serratene triterpenoids, possessing a
seven membered ring C and seven tertiary methyl groups, are a
common fused-pentacyclic triterpene with a double bond between
C-14 and C-15 and oxymethines at both C-3 and C-21; they have
been reported in fern alliances and conifers (Pinaceae) (Conner
et al., 1981, 1984; Fang et al., 1991). Lycernuic acid C, isolated from
and 2.9
inactive.
lM, respectively, while the other compounds were
2. Results and discussion
The whole plant of L. phlegmaria L. (948 g) was extracted with
MeOH and then the greenish extract (204 g) was suspended in
water and defatted with diethyl ether. Purification of the di-ethyl
⇑
Corresponding author at: Laboratory of Medicinal Chemistry, Chulabhorn
Research Institute, Laksi, Bangkok 10210, Thailand. Tel.: +66 (0)2 574 0622; fax:
+66 (0)2 574 2027.
E-mail address: nopporn@cri.or.th (N. Thasana).
ether extract using
a
combination of silica gel column
0031-9422/$ - see front matter Crown Copyright Ó 2012 Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2012.01.006

118
S. Wittayalai et al. / Phytochemistry 76 (2012) 117–123
OH
21
OH
29
21
29
R
2
14
O
1'
14
27
H
3
R
6'
7'
H
O
3
H
24
RO
H
24
HO
R
1
p
1
2
p
1
2
1
1
2
2
OH
21
OH
21
29
29
R
R
2
2
14
14
O
O
OH
H
1
H
1
3
3
HO
HO
O
O
24
H
H
24
HO
R
R
1
1
2
2
1
2
OH
HO
O
H
Fig. 1. Lycopodium serratene triterpenoids (1–9) and abietane diterpene (10).
chromatography and reversed phase C-18 high pressure liquid
chromatography resulted in isolation of four new (5–8) and three
known (1, 2, 9) (Fang et al., 1991; Shi et al., 2005) serratene triterp-
enoids, and an abietane diterpene margocilin (10) (Ara et al., 1990).
Compound 5, obtained as a white powder, possessed the molec-
ular formula C39H58ClO6 on the basis of the positive HRAPCIMS
(micro TOF) at 657.3907 [M+Cl]+ requiring 11° of unsaturation.
Analysis of the 1H and 13C NMR spectroscopic data indicated that
compound 5 was related to 4 (Tables 1 and 2) (Zhou et al., 2003).
In the 13C NMR and DEPT spectra there was 30 carbons for six
methyls, 10 methylenes, five methines, six quaternary carbons,
two oxymethines (d 69.8, C-3 and d 75.3, C-21), and one oxymeth-
ylene (d 68.2, C-24) group indicative of a serratene skeleton, to-
gether with signals of a dihydrocaffeoyl unit (C9H9O3 with 5° of
unsaturation) at C-10 (d 173.3, s), C-20 (d 36.8, t), C-30 (d 31.0, t),
C-40 (d 132.6, s), C-50 (d 116.9, d), C-60 (d 147.3, s), C-70 (d 145.7,
s), C-80 (d 116.6, d), and C-90 (d 119.7, d). Based on the NMR spec-
troscopic analysis of the core structure of compounds 5 and 4, they
differed only in ring A. Analysis of the 13C NMR spectrum of 4, indi-
cated that the signal at d 16.6 (q, C-24) was replaced in 5 by a res-
onance at d 68.2 (t), indicating that an oxymethylene group was
present at C-24. The NOESY experiment of an oxymethylene proton
at C-24 of 5 also correlated to the C-25 methyl group, this being
indicative of a b-configuration for an oxymethylene group. The
orientations of the dihydrocaffeoyl group at C-3 and the hydroxyl
group at C-21 were deduced by comparison of their carbon
(Fig. 2). Due to the difference of the stereochemistry at C-3 and
C-21 of compounds 4 (3b, 21 ) and 5 (3
, 21b), the 13C NMR chem-
a
a
ical shift of C-3 showed a 10.5 ppm difference (d 80.3 for 4 and d
69.8 for 5) as well as a 1H NMR chemical shift difference of H-3
for 0.65 ppm (d 4.70 for 4 and d 4.05 for 5) as shown in Tables 1
and 2, respectively. Additionally the 13C NMR chemical shift of C-
21 of compounds 4 and 5 differed only 2.7 ppm (d 78.0 for 4 and
d 75.3 for 5) and showed a strong effect to the 13C NMR chemical
shift difference of C-29 for 7 ppm (Table 1). The relative configura-
tion of compound 5 was also determined by optical rotation (see
Section 4). Therefore, compound 5 was shown to be a 21b,24-
dihydroxyserrat-14-en-3a-yl dihydrocaffeate as a new serratene
triterpenoid named lycophlegmariol A.
Compound 6, isolated as a yellowish solid, possessed the molec-
ular formula C39H58ClO7 on the basis of the positive HRAPCIMS
(micro TOF) at 673.3877 [M+Cl]+ requiring 11° of unsaturation
and indicated that it had one more oxygen atom comparing with
ompound 5. Analysis of the 1H and 13C NMR spectra indicated that
compound 6 was related to 5 (Tables 1 and 2). The 13C NMR and
DEPT spectra showed 30 carbons for five methyls, 10 methylenes,
five methines, six quaternary carbons, two oxymethines (d 74.3,
C-3 and d 75.7, C-21), and two oxymethylenes (d 64.5, C-24 and
d 70.0, C-29) of a serratene skeleton, together with signals of a
dihydrocaffeoyl unit. Based on the NMR spectroscopic analysis of
core structure of compounds 6 and 5, they differed only in the ring
E. In the 13C NMR spectrum of 5, the resonance at d 22.2 (q, C-29)
was replaced in 6 by a signal at d 70.0 (t), indicating that another
chemical shifts with compound
4 and its 2D NMR spectra

S. Wittayalai et al. / Phytochemistry 76 (2012) 117–123
119
Table 1
13C NMR spectroscopic data of lycopodium triterpenes 3–9.
Position
Lycopodium triterpenes
3a,c
4a,c
5a
6a
7a
8b
9a,d
1
2
3
4
5
6
7
8
38.4(t)
28.7(t)
80.7(d)
38.3(s)
55.8(d)
19.1(t)
45.3(t)
37.4(s)
62.6(d)
38.2(s)
25.5(t)
27.5(t)
57.6(d)
138.6(s)
122.9(d)
24.7(t)
50.1(d)
36.5(s)
37.6(t)
24.3(t)
78.3(d)
39.5(s)
28.2(q)
16.9(q)
16.0(q)
20.1(q)
56.4(t)
13.8(q)
15.5(q)
28.3(q)
172.3(s)
36.9(t)
30.8(t)
131.6(s)
130.0(d)
116.4(d)
157.5(s)
116.4(d)
130.0(d)
38.1(t)
28.4(t)
80.3(d)
37.9(s)
55.5(d)
18.8(t)
45.0(t)
37.1(s)
62.3(d)
37.9(s)
25.2(t)
27.3(t)
57.3(d)
138.4(s)
122.6(d)
24.4(t)
49.8(d)
36.2(s)
37.3(t)
24.0(t)
78.0(d)
39.3(s)
27.9(q)
16.6(q)
15.7(q)
19.8(q)
56.1(t)
13.6(q)
15.2(q)
28.0(q)
172.6(s)
36.7(t)
30.8(t)
132.3(s)
116.7(d)
147.1(s)
145.4(s)
116.3(d)
119.5(d)
33.8(t)
26.6(t)
69.8(d)
42.4(s)
50.0(d)
19.5(t)
45.7(t)
38.4(s)
62.9(d)
37.6(s)
25.4(t)
27.6(t)
57.4(d)
139.1(s)
122.8(d)
24.6(t)
43.8(d)
36.4(s)
31.9(t)
26.5(t)
75.3(d)
38.0(s)
23.4(q)
68.2(t)
16.3(q)
20.0(q)
56.7(t)
13.8(q)
22.2(q)
28.7(q)
173.3(s)
36.8(t)
31.0(t)
132.6(s)
116.9(d)
147.3(s)
145.7(s)
116.6(d)
119.7(d)
34.3(t)
26.8(t)
74.3(d)
43.3(s)
51.7(d)
19.4(t)
45.7(t)
38.4(s)
63.0(d)
37.6(s)
25.4(t)
27.5(t)
57.6(d)
139.1(s)
122.6(d)
24.2(t)
38.2(d)
36.1(s)
31.7(t)
23.5(t)
75.7(d)
40.8(s)
23.0(q)
64.5(t)
16.3(q)
20.2(q)
56.7(t)
14.2(q)
70.0(t)
17.6(q)
172.9(s)
37.0(t)
31.2(t)
132.7(s)
116.9(d)
147.3(s)
145.6(s)
116.6(d)
119.8(d)
37.7(t)
28.7(t)
74.2(d)
43.6(s)
52.0(d)
19.5(t)
45.8(t)
38.5(s)
63.2(d)
37.6(s)
25.8(t)
27.6(t)
57.7(d)
138.7(s)
122.9(d)
24.7(t)
50.2(d)
36.6(s)
34.6(t)
23.7(t)
78.4(d)
39.6(s)
23.1(q)
64.7(t)
16.4(q)
20.2(q)
56.6(t)
13.8(q)
15.5(q)
28.3(q)
167.4(s)
116.1(d)
145.0(d)
126.9(s)
130.7(d)
116.2(d)
161.4(s)
116.2(d)
130.7(d)
38.0(t)
26.3(t)
80.7(d)
38.4(s)
55.7(s)
19.2(t)
44.6(t)
38.1(s)
59.5(d)
38.6(s)
25.6(t)
24.1(t)
60.3(d)
74.8(s)
46.0(t)
19.5(t)
56.8(d)
38.7(s)
38.2(t)
28.0(t)
80.4(d)
43.5(s)
28.7(q)
17.0(q)
16.7(q)
23.1(q)
62.0(t)
16.6(q)
64.6(t)
23.9(q)
172.8(s)
37.0(t)
31.1(t)
132.7(s)
116.9(d)
147.3(s)
145.8(s)
116.6(d)
119.8(d)
38.2(t)
26.3(t)
80.9(d)
38.3(s)
55.7(d)
19.3(t)
44.7(t)
38.2(s)
59.6(d)
38.6(s)
25.8(t)
24.2(t)
60.5(d)
75.0(s)
46.0(t)
19.5(t)
56.2(d)
39.1(s)
39.0(t)
28.5(t)
78.5(d)
39.9(s)
28.1(q)
16.9(q)
16.7(q)
23.4(q)
62.2(t)
16.5(q)
16.7(q)
29.2(q)
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
10
173.0(s)
37.2(t)
31.2(t)
132.8(s)
117.1(d)
147.4(s)
145.8(s)
116.8(d)
119.9(d)
20
30
40
50
60
70
80
90
a
b
c
150 MHz in pyridine-d5.
100 MHz in pyridine-d5.
Zhou et al. (2003).
d
Shi et al. (2005).
oxymethylene group was present at C-29. The NOESY experiment
(Figure in Supplementary data) of the C-29 oxymethylene proton
of 6 correlated to the C-28 methyl group was indicative of a-con-
figuration for the C-29 oxymethylene group. The orientations of
the dihydrocaffeatyl group at C-3 and the hydroxyl group at C-21
were deduced by comparison of their carbon chemical shifts with
compound 5 and its 2D NMR spectra (Figure in Supplementary
data). The stereochemistry at C-3 of compound 6 (3b, 21b) was dif-
related to 3 (Tables 1 and 2) (Zhou et al., 2003). The 13C NMR and
DEPT spectra showed 30 carbons for six methyls, 10 methylenes,
five methines, six quaternary carbons, and two oxymethines (d
74.2, C-3 and d 78.4, C-21) and one oxymethylene (d 64.7, C-24)
of serratene skeleton together with signals of a para-hydroxycinna-
moyl unit (C9H7O2 with 6° of unsaturation) at C-10 (d 167.4, s), C-20
(d 116.1, d), C-30 (d 145.0, d), C-40 (d 126.9, s), C-50 (d 130.7, d), C-60
(d 116.2, d), C-70 (d 161.4, s), C-80 (d 116.2, d), and C-90 (d 130.7, d).
Based on the NMR spectroscopic analysis of core structure of com-
pounds 7 and 3, they differed in ring A. In the 13C NMR of 3, the sig-
nal at d 16.9 (q, C-24) was replaced in 7 by a resonance at d 64.7 (t),
indicating that an oxymethylene group was present at C-24. The
orientations of the cinnamoyl group at C-3 and the hydroxyl group
at C-21 were deduced by comparison of their carbon chemical
shifts with compound 3 and its 2D NMR spectra (Figure in
Supplementary data). The relative configuration of compound 7
was also determined by an optical rotation (see Section 4). There-
ferent from 5 (3a, 21b) and similar to 4 (3b, 21a). The difference in
13C NMR chemical shift of C-3 of 6 as compared to 4 was 6 and
4.5 ppm as compared with 6 (d 80.3 for 4, d 69.8 for 5, and d 74.3
for 6). Also the oxymethylene C-24 showed a significant effect to
the 1H NMR chemical shift of H-3 of compounds 5 and 6 with an
1.7 ppm difference as shown in Table 2 (position 3, d 4.05 for 5,
and d 5.73 for 6). The relative configuration of compound 6 was
also determined by optical rotation (see Section 4). Therefore,
compound 6 was shown to be 21b,24,29-trihydroxyserrat-14-en-
3b-yl dihydrocaffeate as a new serratene triterpenoid named
lycophlegmariol B.
fore, compound 7 was 21
a
,24-dihydroxyserrat-14-en-3b-yl 4-
hydroxycinnamate as
lycophlegmariol C.
a new serratene triterpenoid named
Compound 7, isolated as a white powder, possessed the molec-
ular formula C39H57O5 on the basis of the positive HRAPCIMS
(micro TOF) at 605.419 [M+H]+ requiring 12° of unsaturation. Anal-
ysis of the 1H and 13C NMR spectra indicated that compound 7 was
Compound 8, obtained as a colorless solid, possessed the molec-
ular formula C39H60NaO7 on the basis of the positive HRESIMS
(micro TOF) at 663.4247 [M+Na]+ requiring 10° of unsaturation

Table 2
1H NMR spectroscopic data of lycopodium triterpenes 3–9.
Position
Lycopodium triterpenes
3a,c
4a,c
5a
6a
7a
8b
9a,d
1
2
3
5
1.68, 0.94(m)
1.90, 1.64(m)
4.70(dd, 11.5, 4.7)
0.80(m)
1.68, 0.94(m)
1.90, 1.64(m)
4.70 (dd 11.5, 4.7)
0.80(m)
1.75, 1.52(m)
2.03, 1.85(m)
4.05(br s)
1.50, 1.30(m)
1.96, 1.84(m)
5.73(br s)
1.85, 1.14(m)
1.92, 1.89(m)
5.90(m)
1.68, 1.04(m)
2.00, 1.65(m)
4.58(dd, 11.0, 2.5)
0.93(m)
1.70, 1.06(m)
1.99, 1.73(m)
4.69(dd, 11.3, 4.6)
0.94(m)
1.83(m)
1.59(m)
1.75(m)
6
7
9
1.40, 1.38(m)
1.39, 1.16(m)
0.74(m)
1.74, 1.13(m)
1.90, 1.38(m)
1.83(m)
5.47(br s)
2.15, 2.01(m)
1.38(m)
2.15, 2.01(m)
1.75, 1.64(m)
3.50(dd, 9.1, 6.4)
0.88(s)
0.92(s)
0.82(s)
0.92(s)
2.31, 1.85(ABq,14.3)
0.78(s)
1.08(s)
1.19(s)
2.78(t, 7.5)
3.06(t, 7.5)
7.28(d, 8.4)
7.15(d, 8.4)
7.15(d, 8.4)
7.28(d, 8.4)
1.40, 1.38(m)
1.39, 1.16(m)
0.74(m)
1.74, 1.12(m)
1.90, 1.38(m)
1.83(m)
5.47(br s)
2.15, 2.01(m)
1.38(m)
2.15, 2.01(m)
1.75, 1.64(m)
3.52(dd, 9.3, 6.2)
0.86(s)
0.89(s)
0.78(s)
0.90(s)
2.31, 1.85(ABq,14.3)
0.78(s)
1.10(s)
1.19(s)
2.80(t, 7.5)
3.08(t, 7.5)
7.28(s)
1.54, 1.44(m)
1.37, 1.20(m)
0.95(m)
1.73, 1.02(m)
1.99, 1.03(m)
2.00(m)
1.57, 1.47(m)
1.34, 1.25(m)
0.97(d, 12.0)
1.63, 1.13(m)
2.00, 1.08(m)
2.07(d, 8.8)
5.40(br s)
2.20(d, 16.8), 1.96(m)
2.6(dd, 12.4, 5.2)
1.95, 1.63(m)
1.76, 1.63(m)
4.04(br s)
1.31(s)
4.04, 3.73(ABq,10.8)
0.78(s)
0.76(s)
2.30, 1.76(ABq,14.4)
0.82(s)
1.59, 1.50(m)
1.43, 1.32(m)
1.03(m)
1.74, 1.09(m)
2.40, 2.10(m)
1.84(m)
5.47(br s)
2.16, 2.00(m)
1.37(m)
1.57, 1.32(m)
1.91, 1.85(m)
3.52(br s)
1.43(s)
4.14, 3.81(ABq,10.1)
0.78(s)
0.90(s)
2.31, 1.85(ABq,14.3)
0.78(s)
1.10(s)
1.19(s)
6.88(d, 15.5)
8.10(d, 15.5)
7.65(d, 8.6)
7.15(d, 8.6)
7.15(d, 8.6)
7.65(d, 8.6)
1.65, 1.50(m)
1.85, 1.55(m)
1.68(m)
1.85, 1.25(m)
1.85, 1.68(m)
1.15(m)
2.14, 1.98(m)
1.57, 1.35(m)
1.08(m)
2.14, 1.25(m)
2.00, 1.56(m)
3.73(m)
1.64, 1.50(m)
1.80, 1.49(m)
1.68(m)
1.78, 1.26(m)
1.80, 1.73(m)
1.18(m)
2.03, 1.71(m)
1.63, 1.50(m)
1.06(m)
1.97, 1.21(m)
2.03, 1.60(m)
3.63(dd, 11.2,4.6)
0.89(s)
11
12
13
15
16
17
19
20
21
23
24
25
26
27
28
29
30
20
5.43(br s)
2.05, 1.95(m)
2.15(dd, 11.3, 5.3)
1.98, 1.61(m)
1.93, 1.85(m)
3.66(br s)
1.35(s)
4.47, 4.20(ABq,11.2)
0.80(s)
0.78(s)
2.23, 1.76(ABq,14.8)
0.78(s)
0.93(s)
1.15(s)
2.76(t, 7.5)
3.02(t, 7.5)
7.25(d, 1.8)
–
7.22(d, 8.0)
6.80(dd, 8.0, 1.8)
0.77(s)
0.82(s)
0.85(s)
0.96(s)
0.97(s)
0.91(s)
1.10(s)
1.80, 1.64(ABq, 14.9)
1.44(s)
1.85, 1.51(ABq, 15.1)
1.56(s)
4.63, 3.75(ABq, 11.1)
1.28(s)
2.75(t, 7.4)
3.03(t, 7.4)
7.25(d, 1.9)
–
7.21(d, 8.0)
6.82(dd, 8.0, 1.9)
3.85, 3.65(ABq, 10.8)
0.84(s)
2.87(t, 7.6)
3.08(t, 7.6)
7.27(s)
1.22(s)
1.37(s)
2.86(t, 7.5)
3.13(t, 7.5)
7.35(d, 1.5)
–
7.31(d, 8.1)
6.93(dd, 8.1, 1.5)
30
50
60
–
–
80
7.23(d, 8.1)
6.85(br d, 8.1)
7.21(d, 8.0)
6.83(d, 8.0)
90
a
b
c
600 MHz in pyridine-d5.
400 MHz in pyridine-d5.
Zhou et al. (2003).
d
Shi et al. (2005).

S. Wittayalai et al. / Phytochemistry 76 (2012) 117–123
121
and A549 and inactive against HepG2. Moreover compounds 1 and
8 also showed cytotoxicity in a lower micromolar IC50 range 2.9
and 3.0
whereas compound 6 gave lower cytotoxicity in an order of mag-
nitude (14.7 M); therefore compounds 1 and 8 were ca five times
lM against the MOLT-3 acute lymphoblastic leukemia,
l
more active than compound 6 (Table 3).
3. Conclusions
Fig. 2. Selected 2D NMR spectroscopic data and configurational analysis of
lycophlegmariol A (5).
Investigation of the methanol extract obtained from club mossL.
phlegmaria L. grown in Thailand resulted in the isolation of four new
lycopodium serratene triterpenoids. Their structures were
elucidated on the basis of extensive NMR spectroscopic analysis.
Pentacyclic triterpenoids were established as 21b,29-dihydroxyser-
and indicated that it had two more hydrogen atoms as compared
with compound 6. Analysis of the 1H and 13C NMR spectra indi-
cated that compound 8 was related to lycophlegmarin (9) (Tables
1 and 2) (Shi et al., 2005). The 13C NMR and DEPT spectra showed
30 carbons for six methyls, 11 methylenes, four methines, five
quaternary carbons, one oxyquaternary carbon (d 74.8, C-14),
two oxymethines (d 80.7, C-3 and d 80.4, C-21), and one oxymeth-
ylene (d 64.6, C-29) of a serratene skeleton together with signals of
a dihydrocaffeoyl unit. Based on the NMR analysis of the core
structure of compound 8 and 9, they differed only in ring E. In
the 13C NMR of 9, the resonance at d 29.2 (q, C-29) was replaced
in 8 by a signal at d 64.6 (t), indicating that an oxymethylene group
was present at C-29. NOESY experiment of the C-29 oxymethylene
proton of 8 correlated to the C-28 methyl group and a C-16
rat-14-en-3
trihydroxyserrat-14-en-3b-yl dihydrocaffeate (lycophlegmariol B,
6), 21 ,24-dihydroxyserrat-14-en-3b-yl 4-hydroxycinnamate
(lycophlegmariol C, 7), and 14b,21 ,29-trihydroxyserratan-3b-yl
a-yl dihydrocaffeate (lycophlegmariol A, 5), 21b,24,29-
a
a
dihydrocaffeate (lycophlegmariol D, 8). An abietane-type diterpene,
margocilin (10), was isolated for the first time from a Lycopodium
plant. The isolated lycopodium serratene triterpenoids,
lycophlegmariol B (6), D (8) and compound 1, showed inhibitory
effects against MOLT-3 acute lymphoblastic leukemia (T-lympho-
blast) with IC50 of 14.7, 3.0 and 2.9 lM, respectively.
4. Experimental section
a-methylene proton was indicative of a a-configuration for the
C-29 oxymethylene group. The orientations of the dihydrocaffeoyl
group at C-3 and the hydroxyl group at C-21 were deduced by
comparison of their carbon chemical shifts with compound 9 and
2D NMR (Figure in Supplementary data). The relative configuration
of compound 8 was also determined by optical rotation (see Sec-
4.1. General experimental procedures
Optical rotations were determined on a JASCO DIP1020 polar-
imeter, whereas UV spectra were recorded on a UV 1700 Pharma
Spec Shimadzu spectrophotometer. FTIR spectra were acquired
using a Perkin Elmer Spectrum One using UATR. 1D and 2D NMR
spectra were measured in pyridine-d5 or CD3OD on a Bruker
AVANCE-400 and 600 spectrometers with TMS as internal
standard. High resolution mass spectra were obtained on a Bruker
Daltonics MicroTOF instrument using atmospheric pressure chem-
ical ionization (APCI) in either the positive or negative ion mode.
Preparative HPLC was performed on a Thermo Separation Products
(San Jose, CA) instruments with a UV 6000LP detector using a
Cosmosil column 5C18-Ms-II, 20 ꢀ 250 mm, and Sunfire prep
tion 4). Therefore, compound 8 was shown to be 14b,21
a,29-tri-
hydroxyserrat-3b-yl dihydrocaffeate as new serratane
a
triterpenoid named lycophlegmariol D. Attempt to acetylate tri-
hydroxyserratan 8 failed suggesting it was the 14b-tertiary alcohol
of lycophlemariol D.
The effect of stereochemistry at C-3 and C-21 on the adjacent
positions C-24 and C-29, was analysed and it was concluded that
the difference in stereochemistry at C-3
a and C-3b affected the
13C and 1H NMR chemical shifts of the oxymethylene C-24 as
shown in lycophlegmariols A (5) and B (6); however no data sup-
porting the effect to methyl C-24 was available. On the other hand,
the oxymethylene C-24 also had an effect on the 13C and 1H NMR
chemical shifts of C-3 as shown in lycophlegmariols B (6) and C
C18, 19 ꢀ 250 mm, 5
lm. Sephadex LH-20 were purchased from
GE Healthcare. Thin layer chromatography (TLC) and preparative
thin layer chromatography (PTLC) were carried out on Merck silica
gel (70–230 mesh).
(7). Also the difference in stereochemistry at C-21a and C-21b af-
fected the 13C NMR chemical shift of the methyl and oxymethylene
C-29 moities. Only the oxymethylene C-29 showed a weak effect
4.2. Plant material
on the 13C NMR chemical shift of C-21(
a) as found in lycophlegma-
riol D (8). The variation in 13C and 1H NMR chemical shifts at C-3/C-
24 and C-21/C-29 was the result of the effect of the difference of
stereochemistry at C-3/C-21 and also the oxymethylene group at
C-24/C-29 (Table 4 in Supplementary data).
L. phlegmaria L. (rhizome-stem-sporangia) was collected in
2010 from Satun province, Thailand. L. phlegmaria L. was identified
by Dr. Tripeth Karnchanaphoom, and its voucher specimen (CRI-
Lph-satun-Oct2010) has been deposited at Chulabhorn Research
Institute, Bangkok Thailand.
Serratene triterpenes 1, 6, and 8 were evaluated for their cyto-
toxicities against the cancer cell lines: HuCCA-1 human cholangio-
carcinoma, A549 human lung carcinoma, HepG2 human
hepatocellular liver carcinoma, and MOLT-3 T-lymphoblast (acute
lymphoblastic leukemia). MTT and XTT assays were used to esti-
mate IC50 values, that is, the drug concentration that causes 50%
of cell growth inhibition after 72 h of continuous exposure to the
test molecule. The results obtained are shown in Table 3. Interest-
ingly, compound 1 showed cytotoxicity in a lower micromolar IC50
4.3. Extraction and Isolation
Air-dried whole plants of L. phlegmaria (948 g) were soaked in
MeOH (7 L ꢀ 3) at room temperature for 6 days, and the corre-
sponding extracts were combined and evaporated to dryness. The
crude extract (204 g) was partitioned with H2O (800 mL) and
Et2O (400 mL ꢀ 3). The Et2O fraction (24.8 g) was applied to a silica
gel column (980 g) and eluted with hexane and a gradient of EtOAc
up to 100%, and followed by MeOH to 50% MeOH/EtOAc (each
2500 mL) to afford fractions A to N. Fraction G was purified by
silica gel CC using n-hexane–EtOAc (1:4, v/v) as eluent to give
range 2.62 and 2.42 lM against the HuCCA-1 and HepG2, respec-
tively, but not against the A549 cell line. Lycophlegmariol B (6)
was inactive against most cell lines tested, except the MOLT-3 cell
line. Lycophlegmariol D (8) showed a weak activity with HuCCA-1

122
S. Wittayalai et al. / Phytochemistry 76 (2012) 117–123
Table 3
Cytotoxic activities of serratane triterpenes 1, 6, and 8 in MTT and XTT assays.a
Entry
Compound
Cytotoxic activityc (IC50
HuCCA-1d
[l
M]); mean SD, n = 3)
A549d
HepG2d
MOLT-3e
1
2
3
4
5
1a
2.62
Inactive
Inactive
47.5
0.28
ND
2.42
2.94
14.7
3.0
ND
0.029
6b
Inactive
26.72
0.97
Inactive
Inactive
0.33
8b
Doxorubicin
Etoposide
ND
22.96
ND, not determined.
a
Soluble in DMSO (0.263 mg.mLꢁ1).
b
c
Soluble in DMSO (10 mg.mLꢁ1).
Cytotoxicity was tested against the following cell lines: HuCCA-1 = human cholangiocarcinoma cell line; A549 = human lung carcinoma cell
line; HepG2 = human hepatocellular liver carcinoma cell line; and MOLT-3 = T-lymphoblast (acute lymphoblastic leukemia) cell line.
d
MTT assay.
XTT assay.
e
21b-hydroxy-serrat-14-en-3
a
-yl acetate (2) (65.0 mg). 21b-Hydro-
4.3.5. Lycophlegmarinol C (7)
26
xy-serrat-14-en-3 -ol (1) (126.6 mg) was obtained from fraction I
a
White powder (2.0 mg); [
a]
D + 16.1 (c 0.20, MeOH); Rf 0.35
by on silica gel chromatography eluted with n-hexane–EtOAc (3:7,
v/v) as eluent. Fraction K (1.73 g) was reapplied to a Sephadex LH-
20 column eluted with CH2Cl2/MeOH (1:1, v/v) to give K1 to K3.
Fraction K3 (0.999 g) was purified by HPLC (Sunfire prep C18) to
give 10 fractions (K3a–K3j) and margocilin (10) (2.3 mg, MeOH/
H2O (85:15, v/v) 8.5 mL/min; tR 19.6 min) from fraction K3d. Frac-
tion K3i was purified by HPLC (Cosmosil column 5C18-Ms-II) to
give compounds 7 (2.0 mg, MeOH/H2O (85:15, v/v), 2.7 mL/min;
tR 32.9 min) and 5 (4.3 mg, MeOH/H2O (85:15, v/v) 2.7 mL/min;
tR 27.0 min), whereas fraction K3j was purified by HPLC (Cosmosil
column 5C18-Ms-II) to give compound 9 (2.5 mg, MeOH/H2O
(85:15, v/v) 8.5 mL/min; tR 36.5 min). Fraction M (1.30 g) was ap-
plied to Sephadex LH-20 eluted with CH2Cl2/MeOH (1:1, v/v) to
give fractions M1 to M3. Fraction M3 (0.464 g) was further purified
by HPLC (Cosmosil column 5C18-Ms-II) to give compounds 6
(4.9 mg, 85% MeOH/H2O, 8.0 mL/min; tR 22.3 min) and 8 (28 mg,
MeOH/H2O (85:15, v/v), 8.0 mL/min; tR 34.8 min).
(5% MeOH/CH2Cl2); UV (MeOH) kmax (loge 311 4.72 nm; IR (UATR)
tmax 3355, 2929, 1704, 1604, 1515, 1452, 1385, 1265, 1167, 994,
736 cmꢁ1; for 13C and 1H NMR spectroscopic data are Tables 1
and 2; HRAPCIMS (microTOF) m/z [M+H]+ calcd for C39H57O5:
605.4201, found 605.4190.
4.3.6. Lycophlegmarinol D (8)
27
Colorless solid (28.0 mg); [
a]
D + 6.9 (c 1.6, MeOH); Rf 0.32 (5%
327 3.65, 283.4.16 nm; IR
MeOH/CH2Cl2); UV (MeOH) kmax (log
e
(UATR) tmax 3353, 2926, 1713, 1452, 1367, 1264, 1190, 990,
736 cmꢁ1; for 13C and 1H NMR spectroscopic data are Tables 1
and 2; HRESIMS (microTOF) m/z [M+Na]+ calcd for C39H60NaO7:
663.4231, found 663.4247.
4.3.7. Lycophlegmarin (9)
White powder (2.5 mg); Rf 0.39 (5% MeOH/CH2Cl2); UV (MeOH)
kmax (log
e 323.5 4.29, 284 4.59 nm; IR (UATR) tmax 3393, 2930,
1710, 1604, 1518, 1449, 1365, 1264, 1189, 991, 736 cmꢁ1; for 13C
and 1H NMR spectroscopic data are Tables 1 and 2; HRAPCIMS
(microTOF) m/z [M+Cl]+ calcd for C39H60ClO6: 659.4084, found
659.4065 and 661.4053 (isotopic peak) (Shi et al., 2005).
4.3.1. 21b-Hydroxy-serrat-14-en-3a-ol (1)
27
White powder (126.6 mg); [
(5% MeOH/CH2Cl2); HRAPCIMS (microTOF) m/z [M+Cl]+ calcd for
30H50ClO2: 477.3505, found 477.3497 and 479.3465 (isotopic
a
]
D ꢁ 21.5 (c 0.35, MeOH); Rf 0.51
C
peak) (Fang et al., 1991).
4.3.8. Margocilin (10)
26
Brown solid (2.3 mg); [
a
]
D + 3.2 (c 0.23, MeOH); Rf 0.41 (5%
282 4.68, 232 4.79 nm; IR
(UATR) tmax 3305, 2926, 1652, 1595, 1460, 1375, 1303, 1268,
1177, 1081, 1023, 737 cmꢁ1 1H NMR (600 MHz, C5D5N) ppm
0.96 (s, H18), 1.04 (s, H19), 1.23 (s, H20), 1.25 (d, J = 6.9 Hz, H16),
1.28 (d, J = 6.9 Hz, H17), 1.69 (m, H1 ), 1.8–1.9 (m, H2, H5), 2.25
(d, J = 12.7 Hz, H1b), 2.65 (dd, J = 13.1, 18.0 Hz, H6 ), 2.70 (dd,
J = 4.8, 18.0 Hz, H6b), 3.15 (h, J = 6.7 Hz, H15), 3.33 (dd, J = 4.3,
MeOH/CH2Cl2); UV (MeOH) kmax (log
e
4.3.2. 21b-Hydroxy-serrat-14-en-3
a
-yl acetate (2)
27
White powder (65.0 mg); [
a
]
D ꢁ 0.6 (c 1.1, MeOH); Rf 0.68 (5%
MeOH/CH2Cl2); HRAPCIMS (microTOF) m/z [M]+ calcd for
;
C32H52O3: 484.3911, found 484.3900 (Fang et al., 1991).
a
a
4.3.3. Lycophlegmarinol A (5)
White powder (4.3 mg); [
(MeOH/CH2Cl2 5:95, v/v); UV (MeOH): kmax (log
3.35 nm; IR (UATR) tmax 3400, 2927, 1716, 1603, 1452, 1386,
1264, 1187, 995, 737 cmꢁ1; for 13C and 1H NMR spectroscopic data
are Tables 1 and 2; HRAPCIMS (microTOF) m/z [M+Cl]+ calcd for
28
a]
D ꢁ 13.6 (c 0.45, MeOH); Rf 0.34
320 3.0, 284
11.8 Hz, H3a), 5.50 (br s, OH), 6.64 (s, H11), 7.91 (s, H14); HRAPC-
IMS (microTOF) m/z [M+H]+ calcd for C20H29O3: 317.2111, found
e
317.2113 (Ara et al., 1990).
4.4. Acetylation of Lycophlegmarinol D
C39H58ClO6: 657.3927, found 657.3907 and 659.3939 (isotopic
peak).
A mixture of compound 8 (0.008 g, 0.01 mmol), dimethylamino-
pyridine (0.0023 g, 0.02 mmol) and Et3N (2.6 mL, 0.02 mmol) in
dichloromethane (1 mL) was acetylated with Ac2O (0.01 mL,
0.09 mmol) at 0 °C. The reaction was allowed to room temperature
for 3 h, poured into H2O (20 mL) and extracted with EtOAc
(10 mL ꢀ 3). The organic layers were combined and then evapo-
rated to dryness in vacuo. The residue was purified by PTLC using
4.3.4. Lycophlegmarinol B (6)
Yellowish solid (4.9 mg); Rf 0.29 (MeOH/CH2Cl2 5:95, v/v); UV
(MeOH) kmax (log
e 326 4.77, 289 4.75 nm; IR (UATR) tmax 3355,
2924, 1733, 1517, 1454, 1379, 1266, 1164, 1029, 735 cmꢁ1; for
13C and 1H NMR spectroscopic data are Tables 1 and 2; HRAPCIMS
(microTOF) m/z [M+Cl]+ calcd for C39H58ClO7: 673.3877, found
673.3868 and 675.3834 (isotopic peak).
EtOAc/hexane (3:7, v/v) as eluent to afford lycophlegmariol
D-
21,29,60,70-tetraacetate (11, 0.01 g, 99%). Rf 0.43 (5% MeOH/

S. Wittayalai et al. / Phytochemistry 76 (2012) 117–123
123
CH2Cl2); IR (UATR)
t
max 3385, 2925, 1731, 1593, 1372, 1258, 1237,
1H NMR (400 MHz, C5D5N) ppm 0.79,
Saimanee (Integrated Research Unit), Chulabhorn Research Insti-
tute for conducting the cytotoxicity assays.
1112, 1029, 802, 736 cmꢁ1
;
0.85, 0.90, 1.02, 1.13, 1.28 (each 3H, s, 6 ꢀ methyls on triterpene
nucleus), 0.86 (m, H5), 1.07 (m, H1a,17), 1.15 (m, H13), 1.25 (m,
H11a,19a), 1.40 (m, H6a,16a), 1.56 (ABq, H27a), 1.60 (m,
H7a,16b), 1.65 (m, H20a), 1.67 (m, H1b,2a), 1.68 (m, H6b,9,12a),
1.75 (ABq, H27b), 1.78 (m, H7b), 1.80 (m, H11b,12b), 1.89 (m,
H20b), 1.93 (m, H19b), 1.98 (m, H15a), 2.02 (m, H2a), 2.03, 2.08,
2.25, 2.30 (each 3H, s, 4 ꢀ acetyls of acetylation), 2.12 (H, 15b),
2.70 (t, J = 7.2 Hz, H20), 2.99 (t, J = 7.2 Hz, H30), 4.32 (ABq,
J = 11.6 Hz, H29a), 4.57 (dd, J = 10.8, 4.8 Hz, H21), 4.72 (ABq,
J = 11.6 Hz, H29b), 4.84 (dd, J = 11.6, 4.6 Hz, H3), 7.17 (dd, J = 8.3,
2.0 Hz, H90), 7.29 (d, J = 2.0 Hz, H50), 7.32 (d, J = 8.0 Hz, H80); HRE-
SIMS (microTOF) m/z [M+Na]+ calcd for C47H68NaO11: 831.4654,
found 831.4661.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.phytochem.2012.01.006.
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This work was supported by the Thailand Research Fund (TRF;
RMU5380021 for N.T.) and The Center on Environmental Health,
Toxicology and Management of Chemicals (ETM). The authors are
grateful to Mr. Somchai Pisuitijaroenpong and Mr. Nitirat Chimnoi
for measuring the NMR and MS spectra. Special thanks go to Ms.
Pakamas Intachote (Laboratory of Immunology) and Ms. Busakorn
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